US5310741A - TAN-1251 compounds and their production from penicillium thomii - Google Patents

TAN-1251 compounds and their production from penicillium thomii Download PDF

Info

Publication number
US5310741A
US5310741A US07/674,342 US67434291A US5310741A US 5310741 A US5310741 A US 5310741A US 67434291 A US67434291 A US 67434291A US 5310741 A US5310741 A US 5310741A
Authority
US
United States
Prior art keywords
compound
hydrogen
tan
hydroxy
compound according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US07/674,342
Other languages
English (en)
Inventor
Hideo Shirafuji
Shigetoshi Tsubotani
Takenori Ishimaru
Setsuo Harada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Pharmaceutical Co Ltd
Original Assignee
Takeda Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd filed Critical Takeda Chemical Industries Ltd
Assigned to TAKEDA CHEMICAL INDUSTRIES, LTD. reassignment TAKEDA CHEMICAL INDUSTRIES, LTD. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: HARADA, SETSUO, ISHIMARU, TAKENORI, SHIRAFUJI, HIDEO, TSUBOTANI, SHIGETOSHI
Application granted granted Critical
Publication of US5310741A publication Critical patent/US5310741A/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/10—Spiro-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungi isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/933—Penicillium

Definitions

  • the present invention relates to a novel compound TAN-1251 (hereinafter sometimes referred to briefly as TAN-1251 which, unless otherwise indicated, means a series of TAN-1251 compounds) which is a muscarinic receptor blocking compound of value as a mydriatic or as an antispasmodic/antiulcer agent.
  • the parasympathetic nerve inervates a diversity of peripheral tissues to manifest multi-facted activity.
  • the receptors of acetylcholine, which is its neurotransmitter, are roughly classified into muscarinic receptors sensitive to muscarine which is an alkaloid of Amanita muscaria and nicotinic receptors sensitive to nicotine which is an alkaloid occurring in Nicotiana tabacum.
  • Atropine and scopolamine which are the alkaloids of the belladonna plants, have been utilized for centuries as nonspecific antimuscarinic drugs and are in use even today as mydriatics or antispasmodics [Goodman and Gilman's the Pharmacological Basis of Therapeutics, 7th ed., 130 (1985)].
  • muscarinic receptors can be classified into subtypes, i.e. M1 which has a high affinity for pirenzepine which is an antispasmodic/antiulcer agent and M2 which is low in that affinity [Nature 283, 90 (1980)]. Furthermore, a further ramification of muscarine M2 receptors according to their affinity for AF-DX 116 has been proposed [Life Sciences 38, 1653 (1986) and Clinical and Experimental Pharmacology and Physiology 16, 523 (1989)], and much research is in progress on methoctoramine and other compounds having subtype specificity [Trends in Pharmacological Science 9, 216 (1988)].
  • muscarinic receptor genes have been cloned by genetic engineering techniques [FEBS Letters, 235, 257 (1986)] and so far at least 5 kinds of genes have been reported to exist in man. While the pharmacologic correspondence of them to receptors remains yet to be elucidated, it will not be long before it is clarified [Trends in Pharmacological Science 12, 148 (1989)]. It is expected that muscarinic blockers having novel pharmacologic activity will be developed in this milieu.
  • TAN-1251 is comprised of 4 species which the inventors designated TAN-1251A, B, C and D. Subsequent research revealed that these compounds have the following structures: ##STR2##
  • the inventors of the present invention further conducted degradation and derivatizing experiments using TAN-1251A, B, C and D as starting compounds and examined the biological activity of the degradation products and derivatives. As a result, they found that the following compounds were promising candidates for antispasmodic/antiulcer agents. The finding prompted further study which has resulted in the present invention.
  • the present invention is directed to:
  • R 1 is hydrogen or a hydrocarbon residue which may be substituted;
  • R 2 is oxo or hydrogen and a hydroxy which may be acylated;
  • R 3 is hydrogen or hydroxy which may be acylated; at least one of the dotted lines represents a single bond, or a salt thereof.
  • R 4 is alkyl; X is halogen.
  • An antispasmodic/antiulcer composition containing a compound of the formula (I) or a pharmacologically acceptable salt thereof.
  • An antispasmodic composition containing a compound of the formula (I) or a pharmacologically acceptable salt thereof.
  • An antiulcer composition containing a compound of the formula (I) or a pharmacologically acceptable salt thereof.
  • FIG. 1 shows an UV spectrum of TAN-1251A
  • FIG. 2 shows an IR spectrum of TAN-1251A
  • FIG. 3 shows a 13 C NMR spectrum of TAN-1251A
  • FIG. 4 shows an UV spectrum of TAN-1251B
  • FIG. 5 shows an IR spectrum of TAN-1251B
  • FIG. 6 shows a 13 C NMR spectrum of TAN-1251B.
  • the hydrocarbon group is straight-chain or branched alkyl, alkenyl and alkynyl groups of 1 to 6 carbon atoms.
  • the alkyl group includes, inter alia, methyl, ethyl, propyl, isopropyl, butyl, 1-methylpropyl, 2-methylpropyl, t-butyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, 4-methylpentyl, etc.
  • the alkenyl group includes, inter alia, 2-propyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 3-methyl-2-pentenyl, 4-methyl-3-pentenyl, etc.
  • the alkynyl group includes, inter alia, 2-propyl, 1-methyl-2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-methyl-3-pentynyl, 2-hexynyl and so on.
  • the substituent or substituents thereon include, inter alia, C 3-6 cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.), phenyl which may be substituted (e.g.
  • phenyl o-hydroxyphenyl, m-hydroxyphenyl, p-hydroxyphenyl, etc.
  • hydroxy mercapto
  • C 1-3 alkylthio e.g. methylthio, ethylthio, propylthio, etc.
  • carboxy guanidino, amino, imidazolyl and so on.
  • substituted hydrocarbon groups there may be mentioned cyclohexylmethyl, benzyl, p-hydroxybenzyl, hydroxymethyl, mercaptomethyl, 1-hydroxyethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 3-quanidino-propyl, 4-aminobutyl, 4-imidazolylmethyl, etc.
  • the acyl group includes aromatic acyl groups such as phthaloyl, p-nitrobenzoyl, p-tert-butylbenzoyl, p-tert-butylbenzensulfonyl, benzensulfonyl, toluenesulfonyl, etc.
  • aliphatic acyl groups such as formyl, actyl, propionyl, monochloroacetyl, dichloroacetyl, trichloroacetyl, methanesulfonyl, ethanesulfonyl, trifluoroacetyl, malonyl, succinyl and so on.
  • the salts of the above-mentioned compounds include, inter alia, salts of the conventional kinds, such as the corresponding hydrochlorides, sulfates, phosphates, etc., and the quaternary salts with the nitrogen atom in 4-position, and these salts can be produced by the processes described hereinafter.
  • the microorganism to be employed for the production of TAN-1251A, B, C and/or D in accordance with the present invention may be any microorganism belonging to the genus Penicillium and capable of producing TAN-1251A, B, C and/or D.
  • Penicillium thomii RA-89 which the inventors isolated from the soil in Miyagi Prefecture.
  • the micrological characteristics of this strain are as follows.
  • the RA-89 strain shows good growth on malt extract agar, potato glucose agar and other media, producing abundant conidia.
  • the hyphae are transparent and septate, and conidiophores are adnate from the substrate and aerial mycellia.
  • the conidiophores show little branching but occur singly and are more than 200 ⁇ m long, each having a rough surface with spikes and a swollen end.
  • Detected at the end of the conidiophore are more than 10 phialides in a brush-like formation (penicilli), with tens of conidia arranged in brush-like chains.
  • the conidia are oblong or oval, measuring 3.5 ⁇ 4.0 ⁇ 2.3 ⁇ 2.8 ⁇ m, and have a rough surface.
  • RA-89 strain forms a multiplicity of sclerotia.
  • These sclerotia are amorphous, although primarily oval or spherical, measure about 300 ⁇ m, and although white in an early stage of formation, turn brown with a tinge or orange on aging. Further observation was continued but the sclerotia did not ripen to form ascospores.
  • the strain was cultured on various media at 28° C. for 2 weeks. The results are shown below in Tables 1 and 2.
  • the TAN-1251A, B, C and/or D-producing strains of microorganisms belonging to the genus Penicillium can be caused to mutate by means of ultraviolet light, X-rays, and other radiations, by monospore isolation, or with various chemical mutagens, etc., and even the mutants so obtained and any spontaneous mutants can all be exploited for purposes of the present invention insofar as they cannot be substantially classified as strains of other species in view of the above taxonomical characteristics and as long as they retain the property to produce said particular compound or compounds.
  • the media for culture of such producer strains may be fluid or solid, only provided that they contain nutrients which the strains require and can utilize. However, fluid media are preferred for mass culture.
  • the media should contain sources of carbon and nitrogen which the particular strains used may digest and assimilate, minerals and trace nutrients in appropriate amounts.
  • the useful carbon source includes, inter alia, glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, oils and fats (e.g. soybean oil, lard oil, chicken oil, etc.) n-paraffin and so on.
  • the pH of the medium is preferably controlled near neutral, particularly pH 5.5 ⁇ 7.
  • the incubation temperature and time are preferably about 20° ⁇ 30° C. and about 48 ⁇ 168 hours.
  • the TAN-1251A, B, C and/or D which is produced with the progress of culture can be assayed by the radio receptor assay using a membrane fraction of the rat cerebral cortex as a crude receptor and 3 H-QNB[L-[N-methyl- 3 H]-quinuclidinyl benzilate methyl chloride (Amersham, U.K.) as a radioligand.
  • the production of TAN-1251A, B, C and/or D reaches a maximum in 4 to 5 days of culture.
  • the active substance or substances will be brought into the aqueous layer.
  • an adsorbent resin such as Amberlite XAD-II (Rohm and Haas Co., U.S.A.), Diaion HP-20 (Mitsubishi Kasei, Japan) or Diaion SP-207 (Mitsubishi Kasei, Japan).
  • adsorbent resin For elution of the activity from a column packed with such an adsorbent resin, water or a hydrous solvent such as aqueous methanol, aqueous acetone, etc. can be employed. By concentrating the resulting extract or eluate under reduced pressure, a crude product containing the various species of TAN-1251 is obtained.
  • the eluate thus obtained is concentrated to dryness or freeze-dried, or the concentrate is dissolved in an appropriate solvent such as diethyl ether, ethyl acetate or methanol or a mixture of such solvents and allowed to stand in the cold, whereby the respective species of TAN-1251 can be isolated as powders or crystals.
  • an appropriate solvent such as diethyl ether, ethyl acetate or methanol or a mixture of such solvents
  • HPLC high performance liquid chromatography
  • the stationary phase for HPLC may for example be a reverse-phase column octadecylsilane such as YMC Gel (YMC, Japan) or TSK Gel (Toso, Japan), while the mobile phase may for example be a mixture of acetonitrile or methanol with either an acid, an inorganic salt-containing solution or a buffer solution.
  • each species of TAN-1251 is a basic substance, it can be treated with a suitable mineral acid to give the corresponding salt.
  • the salt can be prepared in the per se known manner. Examples of such salt are the hydrochloride, sulfate, phosphate and so on.
  • UV spectrum Methanol (FIG. 1) Absorption maxima: 265 ⁇ 3 nm ( ⁇ 23,800 ⁇ 3,000) 304 ⁇ 3 nm ( ⁇ 1,600 ⁇ 400, shoulder)
  • IR spectrum KBr disk (FIG. 2); dominant absorptions (wave-number, cm -1 ) 3420, 2980, 2940, 2800, 1720, 1600, 1500, 1450, 1380, 1300, 1250, 1180, 1130, 1030, 920, 890, 830, 780, 750, 690, 620, 530, 510.
  • UV spectrophotometry (214 & 254 nm)
  • IR spectrum KBr disk (FIG. 5); dominant absorptions (wave-number, cm -1 ) 3430, 2940, 2780, 1720, 1600, 1500, 1440, 1380, 1290, 1240, 1170, 1110, 1060, 1000, 920, 880, 850, 820, 580, 520.
  • UV spectrophotometry (214 & 254 nm)
  • UV spectrum Methanol Absorption maxima: 225 ⁇ 3 nm ( ⁇ 7,800 ⁇ 500) 278 ⁇ 3 nm ( ⁇ 1,400 ⁇ 400, shoulder) 285 ⁇ 3 nm ( ⁇ 1,100 ⁇ 300, shoulder)
  • IR spectrum KBr disk; dominant absorptions (wave-number, cm -1 ) 3430, 2950, 2880, 1720, 1680, 1640, 1610, 1510, 1450, 1370, 1320, 1300, 1240, 1170, 1120, 1050, 1000, 860, 840, 810, 790, 730, 630, 510.
  • UV spectrophotometry (214 & 254 nm)
  • IR spectrum KBr disk; dominant absorptions (wave-number, cm -1 ) 3420, 2970, 2940, 2880, 2800, 1720, 1610, 1510, 1450, 1380, 1340, 1300, 1240, 1180, 1150, 1110, 1060, 1020, 1000, 950, 910, 850, 830, 810, 770, 730, 680, 640, 510.
  • UV spectrophotometry (214 & 254 nm)
  • Compounds of general formula (I) can be synthesized from TAN-1251A, B, C and D by conducting the ether bond cleaving reaction with an acid, carbonyl reduction reaction, catalytic double bond reduction, quaternization reaction by alkylation of tertiary amine, hydroxyl acylation reaction, and/or reaction for introduction of a hydrocarbon group into the OH group of phenol in the per se conventional manner.
  • the conversion of Compound 1 to Compound 7, or Compound 2 to Compound 8, is best performed under acidic conditions.
  • the starting compound is dissolved in 0.1 ⁇ 2.0N, preferably 0.2 ⁇ 1.0N, hydrochloric acid or sulfuric acid at a final concentration of 2 ⁇ 50 mg/ml, preferably 5 ⁇ 30 mg/ml, and allowed to react at a temperature of 4° ⁇ 80° C., preferably 10° ⁇ 40° C., for 30 minutes to 2 days, preferably 1 ⁇ 8 hours.
  • the conversion of Compound 1 to Compounds 9 and 10, or Compound 2 to Compounds 11 and 12, is most advantageously carried out using sodium borohydride.
  • the starting compound is dissolved in methanol, ethanol or tetrahydrofuran at a final concentration of 5 ⁇ 100 mg/ml, preferably 10 ⁇ 50 mg/ml, and after addition of 0.2 ⁇ 10 equivalents, preferably 1 ⁇ 5 equivalents, of sodium borohydride, the reaction is carried out at 4° ⁇ 80° C., preferably 10° ⁇ 40° C., for 30 seconds ⁇ 5 hours, preferably 5 minutes to 1 hour.
  • Other reducing agents such as sodium cyanoborohydride, lithium aluminum hydride, etc. can also be employed in lieu of sodium borohydride.
  • the conversion of Compound 1 to Compound 13, or Compound 2 to Compound 14, can be effectively carried out by catalytic reduction.
  • the starting compound is dissolved in methanol or ethanol at a final concentration of 2 ⁇ 50 mg/ml, preferably 5 ⁇ 20 mg/ml and, after addition of a catalytic amount (2 ⁇ 60%, preferably 10 ⁇ 50% by weight) of palladium black, palladium-on-carbon, platinum black or platinum dioxide, the reaction is carried out in a hydrogen gas atmosphere at 4° ⁇ 80° C., preferably 10° ⁇ 40° C., for 1 hour ⁇ 2 days, preferably 2 ⁇ 8 hours.
  • the conversion of Compound 2 to Compound 15 can be carried out most advantageously using methyl iodide.
  • the starting compound is dissolved in methanol, ethanol or propanol at a final concentration of 5 ⁇ 200 mg/ml, preferably 10 ⁇ 100 mg/ml and, after addition of 1 ⁇ 10 equivalents, preferably 1.1 ⁇ 6 equivalents, of methyl iodide, the reaction is carried out at a temperature of 20° ⁇ 100° C., preferably 60° ⁇ 80° C., for 30 minutes to 5 hours, preferably 1 ⁇ 2 hours.
  • the conversion of Compound 2 or 6 to Compound 16 is most effectively carried out using acetic anhydride and pyridine.
  • the starting compound is dissolved in pyridine at a final concentration of 10 ⁇ 1000 mg/ml, preferably 20 ⁇ 500 mg/ml and, after addition of 1 or more equivalents of acetic anhydride, the reaction is conducted at 4° ⁇ 80° C., preferably 10° ⁇ 40° C., for 1 hour ⁇ 3 days, preferably 5 hours ⁇ 2 days.
  • Compound 7 or 8 has a phenolic hydroxyl group(s) and, as such, gives an ether derivative as described hereinafter.
  • an alkyl group into the acidic group of phenol or the like, the following procedures, for instance, are known and can be adequately applied to the production of compounds of the invention.
  • the starting compound is reacted with a diazoalkane (e.g. diazomethane) in a solvent (e.g. ethyl ether, tetrahydrofuran, dioxane, methanol, etc.) at a temperature between about 0° C. and the reflux temperature for a period of about 2 minutes ⁇ 10 hours.
  • a diazoalkane e.g. diazomethane
  • a solvent e.g. ethyl ether, tetrahydrofuran, dioxane, methanol, etc.
  • the starting compound is reacted with an active alkyl halide (e.g. methyl iodide, n-butyl chloride, etc.).
  • an active alkyl halide e.g. methyl iodide, n-butyl chloride, etc.
  • the reaction is conducted in a solvent (e.g. dimethylformamide, dimethylacetamide, etc.) at a temperature of about 0° ⁇ 60° C. for about 2 minutes ⁇ 20 hours.
  • an alkali metal salt e.g. sodium carbonate, potassium carbonate, etc.
  • ammonia triethylamine or the like in the reaction system does not interfere with the reaction.
  • the starting compound is reacted with an alcohol (e.g. methanol, n-butanol, etc.).
  • an alcohol e.g. methanol, n-butanol, etc.
  • this reaction is conducted in a solvent (e.g. dimethylformamide etc.) in the presence of a condensing agent (e.g. dicyclohexylcarbodiimide) at a temperature of about 0° ⁇ 60° C. for about 2 hours ⁇ 2 days.
  • a condensing agent e.g. dicyclohexylcarbodiimide
  • the reaction system may contain an auxiliary condensing agent (e.g. 1-hydroxy-1H-benzotriazole etc.).
  • the hydroxylation of a compound of general formula (I) wherein R 3 H to a compound wherein R 3 is OH can be carried out with the aid of a microorganism belonging to the genus Penicillum.
  • This reaction can be conducted in a medium favoring growth of the microorganism or in the presence of a processed culture broth such as washed cells, immobilized cells and so on.
  • a processed culture broth such as washed cells, immobilized cells and so on.
  • Example 13 A specific example is presented in Example 13, where the microbial transformation of TAN-1251A into TAN-1251B was carried out using Penicillium thomii RA-89.
  • TAN-1251 The biological activity of TAN-1251 is shown below.
  • the assay of activity was carried out by the following two methods.
  • This assay was carried out in accordance with the method of R.F.T. Gilbert et al. [British Journal of Pharmacology 65, 451 (1979)].
  • a Wistar rat male, 8 weeks old, Clea Japan, Inc. was decapitated and the brain was isolated.
  • the cerebral cortex was then separated and using a Teflon homogenizer, the whole cerebral cortex (0.8 ⁇ 1.0 g) was homogenized in 30 ml of 0.32M sucrose solution.
  • the homogenate was centrifuged at 1,000G for 10 minutes and the supernatant was re-centrifuged at 20,000G for 20 minutes.
  • the pellet was used as a crude receptor membrane fraction (P2 fraction).
  • the P2 fraction was suspended in 30 ml of 0.1M sodium potassium phosphate buffer (protein concentration: 0.5 mg/ml) and diluted 50 ⁇ 80-fold with the same buffer. A 200 ⁇ l portion of this dilution was used in the assay.
  • 3 H-QNB (1.63 TBq/mmol, Amersham, U.K.) was added at the level of 0.148 KBq and the sample was added at the same time. The reaction was carried out at room temperature for 60 minutes.
  • the reaction mixture was subjected to rapid filtration through a glass filter (GF/B, Whatman, U.S.A.) to terminate the reaction and after washing with three 300 ⁇ l portions of the same buffer as above, the residual radioactivity on the filter was measured with a liquid scintillation counter.
  • the activity was expressed in the number of units representing the reciprocal of the sample size (ml) required for 50% inhibition or in the corresponding concentration (M), viz. IC 50 .
  • the contraction was recorded through an isotonic transducer (ME-4013, Suruga Electronics, Japan) on a recorder (Rectiholy 8K, Japan Electronics Sanei, Japan).
  • the activity of the same was expressed in ED 50 representing 50% inhibition of the maximal contraction.
  • TAN-1251 is a novel compound and that, as an antimuscarinic agent, it is of value as a therapeutic drug for various diseases such as gastric and duodenal ulcers, spastic pain of the gastrointestinal tract, parkinsonism, etc. or as a mydriatic.
  • TAN-1251 or a salt thereof is administered orally or parenterally as an injectable preparation.
  • the oral dosage for humans is generally 0.05 ⁇ 50 mg/kg/day, preferably 0.1 ⁇ 10 mg/kg/day, and the parenteral dosage is 0.01 ⁇ 10 mg/kg/day, preferably 0.05 ⁇ 5 mg/kg.
  • the dosage form for oral administration includes, inter alia, capsules, tablets, granules, syrups, powders, etc. and may contain, in addition to TAN-1251 or a salt thereof, such additives as various excipients, binders, disintegrators, lubricants, colorants, corrigents, stabilizers and so on.
  • the active compound can be dissolved or suspended in the common diluent (an aqueous or non-aqueous vehicle) and provided in a variety of pertinent dosage forms such as solutions, eye drops, emulsions, suspensions, suppositories and so on.
  • the common diluent an aqueous or non-aqueous vehicle
  • pertinent dosage forms such as solutions, eye drops, emulsions, suspensions, suppositories and so on.
  • additives such as emulsifiers, suspending agents, cosolvents, stabilizers, preservatives, soothing agents, isotonizing agents, buffers, pH adjusting agents, colorants, coating agents and so on.
  • Penicillium thomii RA-89 (IFO-32288, FERM BP-2753) grown on a slant agar medium composed of 24 g potato dextrose broth (Difco, U.S.A.), 20 g agar and 1 l water at 28° C. for 7 days was inoculated into 40 ml of a seed medium (pH 6.5) composed of 2% glucose, 3% maltose, 1% raw soybean flour (SBF), 0.5% corn steep liquor (CSL), 0.25% peptone, 0.15% yeast extract and 0.15% NaCl.
  • the inoculated medium in a 200 ml conical flask was incubated on a rotary shaker at 24° C.
  • a 1000 ml portion of the seed culture obtained in the above manner was transferred to a 200 liter stainless steel tank containing 120 l of a fermentation medium (pH 6.7) containing 5% of glycerol, 2.5% of sucrose, 1% of SBF, 0.5% of peptone, 0.2% of malt extract, 0.1% of yeast extract, 0.2% of ammonium sulfate, 0.5% of calcium carbonate and 0.05% of Aktocol (Takeda Chemical Industries, Ltd., Japan).
  • the inoculated medium was incubated at 24° C., with aeration at a rate of 150 l/min. and agitation at 200 rpm and under an internal pressure of 1 kg/cm 2 for 90 hours.
  • radio receptor assay of TAN-1251 in the culture supernatant its output was 45,000 units.
  • the oil containing TAN-1251A (0.27 g) was combined with the oil containing TAN-1251A (1.8 g) obtained in the same manner and the pooled oil was subjected to preparative HPLC [stationary phase YMC-Pack S-363 I-15 (YMC, Japan), mobile phase 32% acetonitrile/0.01M sodium phosphate (pH 3.0)].
  • the eluate was analyzed by HPLC and the fractions containing TAN-1251A were pooled. A portion (1.4 l) of this solution was concentrated to 0.3 l, adjusted to pH 8.0 and extracted with ethyl acetate (200 ml).
  • the oil containing TAN-1251B (0.49 g) was combined with the oil containing TAN-1251B (0.33 g) obtained in the same manner and the pooled oil was subjected to preparative HPLC [stationary phase YMC-Pack S-363 I-15, mobile phase 25% acetonitrile/0.01M sodium phosphate (pH 3.0)].
  • the eluate was analyzed by HPLC and the fractions containing TAN-1251B were pooled. A portion (500 ml) of this solution was concentrated to 100 ml, adjusted to pH 8.0 and extracted with ethyl acetate (150 ml). The extract was washed with water and concentrated to dryness to provide a powder of TAN-1251B (132 mg).
  • the solution containing TAN-1251A (1.4 l) obtained by preparative HPLC in Example 2 was concentrated and passed through a column of Amberlite IRA-402 (SO 4 2- form, 0.3 l, Rohm and Haas Company, U.S.A.). The effluent and the aqueous wash were chromatographed on Amberlite XAD-II (60 ml) and the activity was eluted with 50% aqueous methanol (240 ml) and 70% aqueous acetone (300 ml). The eluate was concentrated and freeze-dried to provide a white powder of TAN-1251A sulfate (667 mg).
  • TAN-1251B (280 ml) obtained by preparative HPLC in Example 2 was chromatographed using Amberlite IRA-402 (SO 4 2- form, 50 ml) and Amberlite XAD-II (20 ml) and the eluate was concentrated and freeze-dried to provide a white powder of TAN-1251B sulfate (114 mg).
  • UV Absorption maxima in H 2 O: 266 nm ( ⁇ 24,200), 303 nm ( ⁇ 1,900, shoulder)
  • IR KBr disk, dominant absorptions (wave-number, cm -1 ): 3430, 2960, 1720, 1600, 1500, 1450, 1240, 1120, 1000, 830, 620
  • UV Absorption maxima in H 2 O: 264 nm ( ⁇ 25,200), 303 nm ( ⁇ 1,400, shoulder
  • LR KBr disk, dominant absorptions (wave-number, cm -1 ): 3430, 2950, 1720, 1600, 1500, 1450, 1240, 1120, 1010, 620
  • the spores formed on the same slant as used in Example 1 were suspended in 10 ml of water and the whole amount was transferred to a 2 l Sakaguchi flask containing 500 ml of a seed medium and incubated on a reciprocating shaker at 24° C. for 48 hours to give a preculture.
  • One liter portion of the preculture was transferred to a 200 l stainless steel tank containing 100 l of a seed medium (with 0.05% Aktocol added) and incubated at 24° C. with aeration at a rate of 120 l/min. and agitation at 150 rpm under an internal pressure of 1 kg/cm 2 for 48 hours.
  • a 50 l portion of the resulting seed culture was transferred to a 6,000 l stainless steel thank containing 3,600 l of the same fermentation medium as used in Example 1 and incubated at 24° C. with aeration at a rate of 3,600 l/min. and agitation at 200 rpm under an internal pressure of 1 kg/cm 2 for 90 hours.
  • the culture supernatant contained 110,000 units of TAN-1251.
  • the culture broth (3,480 l) was subjected to Oliver filtration using Radiolite (Showa Chemical Industry, Japan) as a filter aid.
  • the filtrate was adjusted to pH 6.5 and passed through a column of Diaion HP-20 (70 l, Mitsubishi Kasei, Japan).
  • the column was washed with water (210 l) and 30% aqueous methanol (210 l) in that order and elution was carried out with 60% acetone/0.01N sulfuric acid (280 l).
  • the eluate was adjusted to pH 4.2 and concentrated to remove the acetone.
  • the resulting aqueous solution (80 l) was adjusted to pH 8.4 and extracted with ethyl acetate (40 l ⁇ 2).
  • the extract was washed with water (25 l ⁇ 2) and concentrated to 5 l and the concentrate was extracted with 0.02N hydrochloric acid (2 l ⁇ 2).
  • the solution was adjusted to pH 3.4, concentrated and subjected to Diaion HP-20 (50 ⁇ 100 mesh, 0.7 l) column chromatography.
  • the column was washed with water (2 l) and 20% aqueous methanol (2 l), followed by elution with 50% aqueous methanol (2.1 l), 60% aqueous acetone (2.1 l) and 70% acetone/0.01N hydrochloric acid (2.1 l) in the order mentioned.
  • the fraction eluted by 70% acetone/0.01N hydrochloric acid was concentrated to 400 ml for removal of the acetone and the concentrate was adjusted to pH 8.2 and extracted with ethyl acetate (200 ml ⁇ 2). The extract was washed with water (150 ml ⁇ 2) and concentrated to dryness. The residue was subjected to silica gel (100 ml; solvent system: chloroform-methanol) column chromatography. The fractions containing TAN-1251A were pooled and concentrated to dryness and the resulting powder was crystallized from ethyl acetate-hexane to provide crystals of TAN-1251A (925 mg).
  • the oil containing TAN-1251C (1.3 g) and the oil containing TAN-1251C obtained in the same manner (4.3 g) were combined and purified by silica gel (300 ml; solvent system: dichloroethane-methanol) column chromatography to give TAN-1251C (5.1 g).
  • the oil containing TAN-1251D (2.0 g) and the oil containing TAN-1251D (8.1 g) obtained in the same manner were combined and purified by silica gel (500 ml; solvent system: dichloroethane-methanol) column chromatography to provide TAN-1251D (7.6 g).
  • UV Absorption maxima in methanol: 264 mm ( ⁇ 22,000), 303 nm ( ⁇ 1,700, shoulder)
  • IR KBr disk, dominant absorptions (wave-number, cm -1 ): 3520, 2970, 2940, 1705, 1610
  • UV Absorption maxima in methanol: 262 nm ( ⁇ 22,500), 303 nm ( ⁇ 1,600, shoulder)
  • UV Absorption maxima in methanol: 265 nm ( ⁇ 29,000), 304 nm( ⁇ 1,900, shoulder)
  • UV spectrophotometry (214 & 254 nm)
  • UV Absorption maxima in methanol: 265 nm ( ⁇ 26,400), 304 nm ( ⁇ 2,000, shoulder)
  • UV Absorption maxima in methanol: 264 nm ( ⁇ 27,000), 303 nm ( ⁇ 1,900, shoulder)
  • UV spectrophotometry (214 & 254 nm)
  • UV Absorption maxima in methanol: 264 nm ( ⁇ 24,600), 303 nm ( ⁇ 1,800, shoulder)
  • UV Absorption maxima in methanol: 264 nm ( ⁇ 21,000), 304 nm ( ⁇ 1,900, shoulder)
  • UV Absorption maxima in methanol: 263 nm ( ⁇ 22,700), 303 nm ( ⁇ 1,600, shoulder)
  • UV Absorption maxima in H 2 O: 266 nm ( ⁇ 22,900), 303 nm ( ⁇ 1,700, shoulder)
  • UV Absorption maxima in methanol: 265 nm ( ⁇ 22,600), 303 nm ( ⁇ 2,000, shoulder)
  • a 200 ml creased Erlenmyer's flask containing 30 ml of a fermentation medium (pH 6.7) composed of 5% glycerol, 2.5% sucrose, 0.5% peptone, 0.2% yeast extract, 0.3% ammonium sulfate and 0.5% calcium carbonate was inoculated with a preculture (inoculum size 1 ml) of Penicillium thomii RA-89 prepared in the same manner as Example 1.
  • a solution of TAN-1251A in dimethyl sulfoxide was diluted 10-fold with methanol, and 0.3 ml of the dilution was added concurrently with inoculation.
  • the flask was incubated on a rotary shaker at 24° C. for 72 hours.
  • the assay of TAN-1251A and B was carried out by HPLC under the Conditions set forth under the heading of Physicochemical Properties. The results are shown in Table 4. It is apparent that approximately 60% of TAN-1251A added was hydroxylated to TAN-1251B.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Botany (AREA)
  • Neurosurgery (AREA)
  • Pain & Pain Management (AREA)
  • Neurology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Saccharide Compounds (AREA)
  • Hydrogenated Pyridines (AREA)
US07/674,342 1990-03-06 1991-03-05 TAN-1251 compounds and their production from penicillium thomii Expired - Fee Related US5310741A (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
JP5574990 1990-03-06
JP2-55749 1990-03-06
JP3-036107 1991-03-01
JP3610791 1991-03-01
JP3-037268 1991-03-04
JP3726891 1991-03-04
PCT/JP1991/000295 WO1991013887A1 (en) 1990-03-06 1991-03-05 Compound tan-1251, its derivatives, their production and use

Publications (1)

Publication Number Publication Date
US5310741A true US5310741A (en) 1994-05-10

Family

ID=27288978

Family Applications (1)

Application Number Title Priority Date Filing Date
US07/674,342 Expired - Fee Related US5310741A (en) 1990-03-06 1991-03-05 TAN-1251 compounds and their production from penicillium thomii

Country Status (9)

Country Link
US (1) US5310741A (de)
EP (1) EP0532752B1 (de)
JP (1) JP3153959B2 (de)
AT (1) ATE174597T1 (de)
CA (1) CA2077672A1 (de)
DE (1) DE69130643T2 (de)
DK (1) DK0532752T3 (de)
ES (1) ES2125235T3 (de)
WO (1) WO1991013887A1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1564284A1 (de) * 2004-02-11 2005-08-17 Urea Casale S.A. Kulturmedium zur Produktion filamentöser Pilze

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04134648A (ja) * 1990-09-26 1992-05-08 Nippon Telegr & Teleph Corp <Ntt> 情報記録部材

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04134648A (ja) * 1990-09-26 1992-05-08 Nippon Telegr & Teleph Corp <Ntt> 情報記録部材

Also Published As

Publication number Publication date
ATE174597T1 (de) 1999-01-15
CA2077672A1 (en) 1991-09-07
JP3153959B2 (ja) 2001-04-09
DE69130643T2 (de) 1999-05-06
DE69130643D1 (de) 1999-01-28
WO1991013887A1 (en) 1991-09-19
EP0532752B1 (de) 1998-12-16
EP0532752A1 (de) 1993-03-24
ES2125235T3 (es) 1999-03-01
DK0532752T3 (da) 1999-06-23
JPH06501916A (ja) 1994-03-03

Similar Documents

Publication Publication Date Title
US4162940A (en) Method for producing Antibiotic C-15003 by culturing nocardia
AU640756B2 (en) 2,8-dioxabicyclo(3,2,1)octane derivatives,their production from cultures of MF 5447 and MF 5466 and their use as anti hyper cholesterolemics
US4421687A (en) Macbecin derivatives
US4421688A (en) Macbecin derivatives
JP2003509508A (ja) ミコフェノール酸およびその誘導体の製造方法
EP0106341B1 (de) Physiologisch wirksame Verbindung P-23924, deren Herstellung und Verwendung
US4530790A (en) Cholecystokinin antagonists
US5310741A (en) TAN-1251 compounds and their production from penicillium thomii
EP0080695B1 (de) Physiologisch wirksame Verbindung FA-5898, deren Derivate, Herstellung und diese Verbindungen enthaltende antidiabetische Mittel
GB2106111A (en) Macbecin derivatives and their production
US5523219A (en) Enzymatic hydrolysis method for the preparation of C-10 hydroxyl-bearing taxanes and enzymatic esterification method for the preparation of C-10 acyloxy-bearing
US5284758A (en) Process for forming cholesterol lowering compound using pseudodiplodia sp.
CA1107212A (en) Antibiotic c-15003
GB2174696A (en) Cyclic adenosine-3&#39;,5&#39;-monophosphate phosphodiesterase inhibitors
US4618581A (en) Clavine-producing strain, a process for the preparation thereof as well as a microbiological process for producing clavine alkaloids
GB2261375A (en) Inhibitors of farnesyl protein transferase as anti-cancer agents
JPS5831992A (ja) 新規生理活性物質ｋ−４およびその製造法
US5260215A (en) Fungal microorganism ATCC 74167 capable of producing cholesterol lowering compounds
Wong et al. Microbial hydroxylation of (Z)-2-benzylidene-1-azabicyclo [2.2. 2] octan-3-one
USRE34414E (en) Physiologically active substance P-23924, its production and use
US4803275A (en) 2-oxo-5-methyl-6-[4-heptene]-2H-pyrano[3,2-c]pyridine
US7060821B2 (en) Osteoclast differentiation inhibitors
EP0629184A1 (de) Tetralinderivate als hmg-coa reduktase inhibitoren
US4287302A (en) Microbiological modification of antibiotic A23187 esters
JP2786267B2 (ja) 新規物質ｄｃ１１６

Legal Events

Date Code Title Description
AS Assignment

Owner name: TAKEDA CHEMICAL INDUSTRIES, LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:SHIRAFUJI, HIDEO;TSUBOTANI, SHIGETOSHI;ISHIMARU, TAKENORI;AND OTHERS;REEL/FRAME:006250/0221

Effective date: 19910416

FEPP Fee payment procedure

Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

FPAY Fee payment

Year of fee payment: 4

FEPP Fee payment procedure

Free format text: PAYER NUMBER DE-ASSIGNED (ORIGINAL EVENT CODE: RMPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

REMI Maintenance fee reminder mailed
LAPS Lapse for failure to pay maintenance fees
STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20020510